Methods to identify compounds affecting mitochondria

ABSTRACT

The present invention relates to methods for identifying a compound capable of modulating mitochondrial function, comprising contacting a eukaryotic cell with one or more candidate compounds, and detecting a change in the mitochondrial redox state of the cell. The methods further relates to such methods wherein endogenous fluorescence of the cell mitochondria is indicative of a change of redox state.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a continuation of U.S. Ser. No. 09/060,774filed on Apr. 15, 1998 (now U.S. Pat. No. 6,183,948). The disclosure ofsaid U.S. Pat. No. 6,183,948 is incorporated herein by reference.

FIELD OF THE INVENTION

In one aspect, the present invention relates to methods for screeningfor compounds that modulate mitochondrial function by affectingmitochondrial redox potential. Methods of the invention also can be usedto test for mitochondrial fitness.

BACKGROUND OF THE INVENTION

Nearly every cell of the body contains organelles called mitochondriawhich produce most of the energy used by the body. Certain cells withhigh metabolic rates, such as heart muscle cells, may contain thousandsof mitochondria.

Energy derived from the utilization of substrates is required in orderto maintain the non-equilibrium state necessary to carry out the basicfunctions of the cell (e.g. contraction, secretion, electricalpropagation, ion pumping, cell division). There are three major stepsinvolved in cellular energy production. First, large food molecules arebroken down into smaller units. Polysaccharides are converted to simplesugars, fat is converted to fatty acids and proteins are converted toamino acids. Second, multiple pathways convert these three differentmolecules into a common precursor, acetyl CoA, for further oxidation bythe mitochondria. Third, the Krebs cycle (a.k.a. the citric acid ortricarboxylic acid cycle) and oxidative phosphorylation convert acetylCoA into ATP, the energy storage molecule directly utilized by theenergy consuming reactions in the cell.

Mitochondrial substrate oxidation is a multi-step process that includesa series of reactions which transfer electrons from the initialsubstrate to nicotinamide adenine dinucleotide (NAD+) to produce NADHwhich is then reoxidized by passing electrons through the electrontransport chain to the ultimate electron acceptor oxygen. In the processof electron transfer, protons (H+) are pumped out of the mitochondrialmatrix (an intracellular aqueous compartment bounded by the inner andouter mitochondrial membranes between it and the cell's cytoplasm)across the mitochondrial inner membrane (IMM) resulting in theestablishment of a proton gradient. This proton concentration gradient,together with the large membrane voltage generated by the active chargemovement across the IMM, provide the driving force for proton movementback into the matrix (the protonmotive force) which will be utilized bya specific IMM protein (the mitochondrial ATP synthase) to convert ADP(adenosine diphosphate) to ATP (adenosine triphosphate). ATP thusproduced is transported out of the mitochondria and is available toperform the work required by the cell.

The relative impermeability of the IMM to ion leak and the presence ofenergy conserving pumps and exchangers in the membrane allows for theefficient utilization of the protonmotive force for cellular energyproduction rather than expending it for reestablishing the IMM gradient.Despite this requirement for maintaining a high resistance membrane, theIMM contains a number of energy dissipating high conductance pathwaysfor ion and/or solute movement. The physiological role of some of thesepathways is apparent, for example, the pyruvate transporter is requiredto import substrate into the matrix for oxidative phosphorylation;however, in other cases, the physiological importance of IMM ionconducting pathways is still unknown. A well known example is themitochondrial megachannel (MMC) or permeability transition pore (PTP).When activated, this large non-selective channel rapidly de-energizesthe mitochondrion and has been implicated in several pathophysiologicalstates. Other known high conductance pathways include the calciumuniport, the mitochondrial inner membrane anion channel, themitochondrial uncoupling protein of brown fat mitochondria, and themitochondrial ATP-sensitive potassium channel. A number of electrogenic(e.g., the adenine nucleotide transporter, the glutamate-aspartatetransporter, the Na—Ca exchanger), proton-compensated electroneutral(e.g., the glutamate, pyruvate, and malate-citrate transporters) andelectroneutral (e.g., the malate-phosphate, malate-ketoglutarate,carnitine, ornithine, and neutral amino acid transporters) are alsopresent in the IMM and may influence the mitochondrial energy state.Although some of these channels and transporters have been well studiedin isolated mitochondria, for lack of a useful index of mitochondrialactivity in intact cells, much less is known about their regulation orsensitivity to pharmacological agents in intact cells.

The mitochondria are essential for efficiently providing ATP forcarrying out the myriad functions of the cell, particularly in tissueswith a high energy demand, such as muscle and brain. Consequently,defects in mitochondrial energy metabolism are usually associated withsignificant functional deficits or death. The pathophysiologies relatedto mitochondrial dysfunction can be either primary or secondary. Inprimary mitochondrial diseases, a genetic defect (either inborn oracquired) in a mitochondrial protein may lead to the incorrect assemblyor catalytic activity of the protein, thus disrupting or impairing theentire biochemical pathway. Secondary mitochondrial disorders may arisefrom the accumulation of toxic products within the cell (includingoxygen free radicals), the accumulation of inhibitory metabolites, orlack of cofactors required for mitochondrial metabolism.

Mitochondrial cytopathies of differing origin often lead to similarclinical symptoms. Commonly the disorders are first expressed in themost metabolically active tissues. They may present as muscle weaknessand fatigue, mild muscle ache, or severe (and sometimes lethal) lacticacidosis during exercise. In many cases, these muscle deficiencies arealso associated with central nervous system disorders, referred to asmitochondrial encephalomyopathies (e.g, KSS, Kearnes-Sayre syndrome;MERRF, myoclonus epilepsy with ragged red fibres; MELAS, mitochondrialencephalomyopathy/lactic acidosis/stroke). These disorders may arisefrom point mutations in or deletions of large segments of mitochondrialDNA. In many cases, the specific enzyme affected is known (e.g. pyruvatedehydrogenase deficiency). Similarly, defective nuclear encoded proteinsinvolved in mitochondrial metabolism or drugs interfering withrespiration (e.g., AZT or Adriamycin) can also lead to mitochondrialcytopathies.

Mitochondrial cytopathies can be classified by the site of the defect inmitochondrial oxidation. Defects in substrate transport (e.g. carnitineor carnitine-palmitoyl-transferase deficiencies), substrate metabolism(e.g. deficiencies in pyruvate dehydrogenase, pyruvate carboxylase,fatty acid oxidation, or organic acid metabolism), Krebs cycle activity(e.g. defects in oxoglutarate dehydrogenase or fumarase), therespiratory chain (NADH-Q reductase or cytochrome deficiencies), orenergy coupling (ATP synthase defect, mitochondrial uncouplingdiseases).

Cumulative alterations in mitochondrial metabolism have been suggestedas an underlying cause of diseases associated with aging, includingAlzheimer's and diabetes mellitus. Furthermore, mitochondria are thesite of initiation of programmed cell death (apoptosis) and probably arethe key factor in determining whether or not a cell will recover from anischemic insult or proceed to necrosis.

Thus, mitochondria are central to the survival and function of the cellunder normal conditions and play a major role in adapting toenvironmental stress.

Thus, it would be desirable to have a method of studying mitochondrialfunction as well as methods of assessing the effect of differentchemicals on mitochondrial function. It would be particularly desirableto identify compounds that selectively modulate mitochondrial function.It would be also useful to detect compounds that affect mitochondrialK_(ATP) channels.

SUMMARY OF THE INVENTION

The present invention relates to methods of assaying for mitochondrialfunction and more particularly to methods of identifying compounds thatselectively modulate mitochondrial function. In one aspect, the presentinvention relates to methods of detecting compounds that can positivelyimpact mitochondrial function and increase cell energy output. In arelated aspect, the invention relates to methods of detecting compoundsthat can decrease mitochondrial function in diseased cells. The presentinvention has a variety of useful applications including use in screensto detect compounds that can enhance overall health and fitness.

More particularly, the invention includes methods for detecting theeffects of agents acting on mitochondrial metabolism in intact cells byutilizing endogenous redox potential sensitive fluorophores located inthe mitochondria. The methods of the invention can enable low cost highthroughput screening of compounds which modify the functional state ofmitochondria for therapeutic applications. Methods of the invention areapplicable to the discovery of agents which modify the activity of anyof the steps in energy metabolism. For instance, an exemplary andpreferred application detects mitochondrially active agents in intactcardiac cells.

In general, the methods of the present invention are useful fordetecting drugs that alter mitochondrial function. For example, in oneaspect, the invention provides a drug detection assay by measuringendogenous fluorescence in intact cells. In a normal oxygenated medium,the mitochondrial matrix is significantly reduced. Drugs that decreasethe membrane potential across the inner mitochondrial membrane causeoxidation of the matrix, which is detected as a change in endogenousfluorescence by the methods of the present invention. Accordingly, themethods of the present invention are well-suited to detect compoundsthat can selectively enhance or decrease mitochondrial function.

In the present methods, cells are cultured and illuminated atwavelengths suitable to excite endogenous fluorescence. Preferably thefluorescence is due to changes in the redox state of endogenousmolecules located in the mitochondria. These endogenous moleculesfunction as reporters of mitochondrial oxidation state. Preferredmolecules include endogenous proteins that comprise fluorescentmolecules such as a flavin moiety, or endogenous fluorescent moleculessuch as NAD.

One aspect of the invention relates to a method for identifying acompound capable of modulating mitochondrial function comprisingcontacting a eukaryotic cell with one or more candidate compounds anddetecting a change in the mitochondrial redox state. Preferably,endogenous fluorescence of the cell mitochondria is indicative of achange of redox state. The change in the redox state is an increase ordecrease in the state of the mitochondria oxidation. That change istypically related to a suitable control assay as described below.

In certain preferred methods of the present invention, the fluorescenceis measured of a nicotinamide adenine dinucleotide (NAD) or a flavinadenine dinucleotide (FAD) moiety, such as a protein comprising a linkedflavin adenine dinucleotide (FAD) moiety. In embodiments comprising sucha FAD-linked protein, preferably the FAD-linked protein is linked to aprotein component of a mitochondrial redox pathway.

In preferred methods, detection of the mitochondrial redox state furthercomprises measuring a change in fluorescence of an NAD molecule orFAD-linked enzyme, and correlating that change to a control assaycomprising a mitochondrial oxidizing or reducing agent. Illustrativeoxidizing agents include dinitrophenol, and illustrative reducing agentsinclude cyanide.

In certain embodiments of the present invention, the cell is contactedwith a plurality of candidate compounds or a library of candidatecompounds.

In certain embodiments the steps of contacting a eukaryotic cell withone or more compounds and detecting the change in the mitochondrialredox state of the cell are performed a number of times substantiallysimultaneously. These steps can be performed e.g. in a multi-well plate.

The eukaryotic cell used in certain methods of the present inventioncomprises a cardiac cell or a precursor cell thereof. In certainpreferred embodiments, the eukaryotic cell is a cardiac cell orprecursor cell thereof that is immortalized. In other preferred methods,the cardiac cell or precursor cell thereof is a primary cell. The cellmay comprise a ventricular myocyte or a skeletal myoblast.

The present invention relates to methods for detecting many differenttypes of drugs capable of modulating mitochondrial function. The presentinvention also relates to methods wherein the candidate compoundactivates a mitochbndrial K_(ATP) channel. Further, it relates tomethods of assaying the activity of mitoK_(ATP) channels usingfluorescence methods. In certain preferred embodiments, the compounddoes not substantially activate a sarcolemmal K_(ATP) channel.

In certain embodiments of the present methods, the cell is contactedwith the candidate compound(s) in vitro. In other embodiments, the cellis contacted with the candidate compound(s) in vivo. In yet otherembodiments the cell is a tissue and the tissue is treated with thecandidate compound ex vivo.

The present invention further relates to a method for detecting acompound capable of modulating mitochondrial redox potential, the methodcomprising:

a) providing a population of eukaryotic cells;

b) contacting a first portion of the cells with one or more candidatecompounds;

c) contacting a second portion of the cells with a known mitochondrialoxidizing or reducing agent; and

d) measuring a difference between mitochondrial fluorescence produced insteps b) and c).

It will be appreciated that in cases where the mitochondrialfluoresecence of a known oxidizing or reducing agent is known, it willnot always be necessary to perform step c), above.

In particular embodiments of this method, the cells are ventricularcells and the method further comprises measuring mitochondrial K_(ATP)ion channel currents in those cells. In some embodiments of this method,the compound activates a mitochondrial K_(ATP) ion channel in the cells.In other embodiments, the method further comprises measuring sarcolemmalK_(ATP) ion channel currents in the cells. In some examples of suchmethods, the drug does not substantially activate the sarcolemmalK_(ATP) ion channel currents at comparable concentrations.

In certain embodiments of the methods, the mitochondrial fluorescence isactivated by a light at a wavelength of from about 250 to about 650 nm.In these embodiments, the step of detecting or measuring is accomplishedby fluorescence microscopy.

The methods of the present invention are applicable to nearly anyeukaryotic cell. Preferred cells comprise detectable mitochondrialfluorescence. For example, such cells will often include cells fromhighly energetic tissues such as muscle and particularly cardiac andskeletal muscle cells. In addition certain rapidly dividing cells canalso be used such as cancer cells (primary or cultured cell line) andimmature cells (e.g., hemapoeitic cells). However, in preferredembodiments, the eukaryotic cells are selected from the group consistingof H9C2 (rat ventricular myocyte-derived cell line), AT-1, HL-1 (atrialtumor derived cell line) and C212 (murine skeletal muscle-derived cellline) cells.

Preferably, the methods identify a candidate compound drug thatmodulates mitochondrial oxidation (e.g. mitochondrial flavoproteinoxidation) by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%or 100%, in a mitochondrial redox assay of the invention relative to acontrol (i.e. the same assay where the candidate compound has not beenexposed to the test cells). The EC₅₀ of identified candidate compoundsis preferably no more than about 10 μM in a standardwhole-cell-patch-clamp assay.

The invention further relates to a method of detecting the activity of amitochondrial ion channel or mitochondrial transporter comprisingcontacting a eukaryotic cell with one or more candidate compounds anddetecting a change in the mitochondrial redox state as indicative of theactivity of the ion channel or transporter.

The invention also further relates to drug compounds obtained by theabove-described method.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1(A) and (B) are graphs showing the effect of diazoxide onflavoprotein fluorescence and I_(K ATP), respectively.

FIG. 2(A) is a graph showing the effect of 5-HD on the oxidative effectof diazoxide on flavoprotein and FIG. 2(B) is a graph showing effect of5-HD.

FIG. 3 is a graph showing pooled data for fluorescence.

FIG. 4 shows pooled data for I_(KATP).

FIG. 5 is a graph showing a dose response curve for diazoxide.

FIGS. 6(A) and (B) show the effect of pinacidil on flavoproteinfluorescence and I_(KATP), respectively.

FIGS. 7(A) and (B) are graphs showing summarized data for percentage offlavoprotein oxidation and I_(KATP), respectively.

FIG. 8 are confocal images showing (A) a cell at baseline and (B) a cellafter TMRE loading of the same cell. (Control), after 3 minute exposureto diazoxide (Diazo), after exposure to dinitrophenol (DNP) and cyanide(CN).

FIG. 9 is a graph of pooled data showing protection of rabbitventricular myocytes from ischemia by diazoxide.

DETAILED DESCRIPTION OF THE INVENTION

We have found methods of screening compounds that modulate the functionof the mitochondria, particularly affecting the redox state of thatorganelle. As described above, the methods are well-suited to detectcompounds capable of modulating mitochondrial function. One method ofthe present invention for detecting a compound capable of modulatingmitochondrial function comprises contacting a eukaryotic cell with oneor more candidate compounds and detecting a change in the mitochondrialredox state.

The present methods of identifying compounds that modulate mitochondrialfunction are more easily performed than previously known methods.Furthermore, the present methods involve minimal cost or expense toperform, as compared to other methods.

The term “candidate compound” as used herein refers to any chemicalcompound that can be added to a eukaryotic cell, and may comprise acompound that exists naturally within the cell or is exogenous to thecell. The compounds include native compounds or synthetic compounds, andderivatives thereof. These compounds may also be referred to herein as“compound to be tested” or “compounds of interest”.

The compounds to be identified modulate the function of mitochondriapreferably by eliciting a change in the redox state of the mitochondria.The term “redox state” means the degree or level of oxidation orreduction of the matrix of the mitochondria at any given time. In anormal oxygenated environment, the mitochondrial matrix is substantiallyin a reduced state. The redox state of the mitochondria can change asthe result of many factors. As described above, certain disease statescan alter the redox state of the mitochondria. In addition, certaincompounds, or drugs, may alter the redox state of the mitochondria. Forexample, drugs that decrease the membrane potential across the innermitochondrial membrane cause oxidation of the matrix. For example, theopening of any potassium-selective ion channels in the innermitochondrial membrane would tend to dissipate the membrane potentialestablished by the proton pump. (see e.g., Garlid, K. D., Biochimica etBiophysica Acta, 1996, 1275: 123-126). Such dissipation accelerateselectron transfer by the respiratory chain, and leads to net oxidationof the mitochondrial matrix.

Other processes may also change the redox state, e.g., changes inmetabolism, glycolysis, oxidative phosphorylation, Kreb cycle, etc.Thus, the mitochondrial redox state is an important indicator ofmitochondrial function. Compounds that can affect the degree ofmitochondria oxidation are generally referred to herein as “mitochondriamodulating compounds” or other similar term.

The detection of the redox state of mitochondria can be accomplished bymethods known in the art. Certain components within the mitochondriafluoresce in response to changes in the redox state of the mitochondria.Endogenous fluorescent molecules in the mitochondria comprisenicotinamide adenine dinucleotide (NAD/NADH), or molecules that comprisea flavin adenine dinucleotide (FAD) moiety. The fluorescence of aFAD-containing molecule increases as the oxidation state inside themitochondria increases, and decreases as the interior becomes reduced.The fluorescence of NAD decreases as the oxidation state inside themitochondria decreases, and increases as the interior becomes reduced.Thus, the degree of fluorescence, or change in fluorescence, provides ameasurement of the redox state of the organelle. (Chance, B., et al.,Am. J. Physiol., 1972, 223 :207-218; Hajnoczky, G., et al., Cell, 1995,82: 415-424). Other methods for detecting a change in the mitochondrialredox state of the cell also could be employed.

The eukaryotic cells used in the methods of the present invention aresometimes referred to herein as “test cells” or “cells to be tested.”

The steps in the methods of the present invention can be performed inany one of a number of ways. For example, a population of cells to betested can be divided into portions and the portions placed in the wellsof multi-well plates, test tubes or eppendorf tubes, or other suchholders as known in the art. Furthermore, the contacting of the cellswith one or more candidate compounds can be performed in numerous ways.

In one method for identifying a mitochondrial modulating compound, atleast one portion of the cells to be tested are exposed to at least onecandidate compound, while another portion of the cells acts as acontrol, without exposure to any compounds. If desired, another portionof the cells is exposed to a compound that is known to either reduce oroxidize the mitochondria. The numbers of portions of cells to be testedwill depend on the number of candidate compounds. The mitochondria redoxstate of the cells is then measured.

In assays that measure fluorescence of a molecule as an indicator of theredox state of the mitochondria, the cells are exposed to an appropriatewavelength of light, selected to excite the fluorescent molecule. One ofordinary skill in the art can readily select the appropriate wavelengthand source of light by known methods in conjunction with the teachingsdescribed herein. The amount of fluorescence of the cells exposed to thecandidate compound is compared to the fluorescence of the control cells.The difference in fluorescence is correlated to the change in the redoxstate of the mitochondria produced by exposure of the cells to thecandidate compound as compared to the control cells.

In other embodiments, the fluorescence of the test cells is measuredprior to addition of one or more of the candidate compounds. In thisembodiment, fluorescence is then measured again after the cells arecontacted with the candidate compounds. Alternatively, fluorescence ismeasured during the course of contacting the cells, so that the changein fluorescence can be measured simultaneously with the contact. Thechange in fluorescence is correlated to a change in the redox state ofthe mitochondria in response to the candidate compounds.

The cells may be exposed to one or more candidate compoundssequentially. That is, if the cells are divided into portions, eachportion of test cells may be contacted with one compound and thefluorescence in response to that compound measured. The cells may thenbe washed by methods known in the art and then contacted with anothercandidate compound and fluorescence in response to that compoundmeasured. These washing, contact and measuring steps can be repeatednumerous times depending on the number of compounds to be tested.Alternatively, the washing step can be eliminated and the compoundsadded sequentially.

The cells can be contacted with a library of candidate compounds. Forexample, the conditions are optimized to provide a measurable andreversible change in fluorescence in response to known compounds, e.g.,diazoxide and dinitrophenol (DNP). Then each compound in the library isplaced in a well in a multi-well plate containing a portion of cells tobe tested. The compounds that produce the desired response, e.g.,increase in oxidation, can be selected based upon the degree offluorescence, e.g., increase in fluorescence produced by the cells.

High-throughput screening involves the testing of a range of differentchemical entities in biochemical assays. Any of the methods ofidentifying mitochondrial modulating compounds described herein can beused for high-throughput screening assays. The “hits”, i.e., compoundsthat produce the desired response, found by any of the methods, can thenbe further characterized according to methods known in the art, e.g.,using cellular physiology and imaging methods.

In other embodiments, after the cells are contacted with candidatecompounds, the cells are further exposed to conditions that create orsimulate a disease state. For example, to test a compound forcardioprotective properties the cells are exposed to simulated ischemiaand cell injury subsequent to treatment with one or more candidatecompounds. Such models are known in the art. An exemplary model isfurther described below and in the examples that follow. In such amethod, the degree of prevention of the disease state is determined bycomparing the fluorescence of the cells treated with candidate compoundswith the fluorescence of untreated, diseased cells. Candidate compoundsare selected if they prevent the degree of fluorescence exhibited by thecontrol cells, i.e., the change in redox state caused by the disease.

In another example, the test cells either have a disease state or normaltest cells are subjected to conditions that create a disease state. Thediseased cells will have a certain degree of fluorescence based on theredox state created by the disease. The cells are then contacted withcandidate compounds and the change in fluorescence in response to thecompounds is measured. The compounds that reverse the fluorescence,i.e., the redox state of the cells, due to the disease can be selected.

As discussed above, the fluorescent molecule may comprise a proteinwhich has a flavin adenine dinucleotide (FAD), or the fluorescentmolecule may comprise nicotinamide adenine dinucleotide (NAD). Inembodiments comprising a FAD-linked protein, preferably the FAD-linkedprotein is an enzyme component of a mitochondrial redox pathway. Thewavelength used to excite the fluorescent molecule is preferably withinfrom about 250 nm to about 650 nm, and most preferably at about 488 nmwhen the fluorescent molecule contains a FAD moiety. When the moleculecontains a NAD moiety, the wavelength is suitably within from about 250to about 450 nm, and more preferably from about 320 to about 400 nm,with about 360 nm being particularly preferred.

Fluorescence can be recorded using presently known methods andtechnology, e.g., standard or specialty fluorescent plate readers. Thefluorescent images can be analyzed by computer using software designedfor such imaging, e.g., ImageTool (Univ. of Texas Health SciencesCenter, San Antonio). Fluorescence of NAD moiety can be detectedaccording to methods known in the art, particularly those described inO'Rourke, B., et al., Science, 1994, Vol. 265, pp. 962-6. Fluorescencecan also be detected by methods, such as, but not limited to fluorescentmicroscopy, photometry, and photographic film.

As described above, the method may comprise measuring fluorescence of anNAD molecule or FAD-linked enzyme as a result of adding a candidatecompound of interest and comparing that fluorescence to the fluorescencein a control assay that comprises a known mitochondrial oxidizing orreducing agent. Useful oxidizing or reducing agents are known in theart. Preferred oxidizing agents comprise dinitrophenol (DNP) anddiazoxide. DNP is a protonophore which uncouples respiration from ATPsynthesis and collapses the mitochondrial potential and inducesoxidation in the mitochondria. A preferred reducing agent comprisescyanide. Cyanide inhibits cytochrome oxidase and thus stops electrontransfer. Other oxidizing and reducing agents can be readily selectedfor use in the control in accordance with the present disclosure.

The known mitochondrial oxidizing or reducing agent can be added to thetest cells subsequent to addition of the candidate compounds. In such anassay, the candidate compounds may or may not be washed from the testcells, depending on the assay protocol. The compounds, however, need notbe washed from the cells for this assay to work. Thus, if desired, onepopulation of cells can be used to test a number of candidate compounds.

Any eukaryotic cell can be used in the identifying methods. Preferablythe cell contains numerous mitochondria, and a sufficient number toenable analysis in accordance with the methods of the invention. Inpreferred embodiment, the cell comprises a cardiac cell or a precursorcell thereof. These cells may be immortalized or a primary cell.Examples of preferred cells comprise a ventricular myocyte or a skeletalmyoblast. For high-throughput screening assays, as described below, thecells should be easy to produce and have good survival rates in culture.Such cell lines can readily be selected by one of ordinary skill in theart. Examples of useful cell lines include H9c2 cells, AT-1 cells andC2C12 skeletal myoblasts. The cells preferably will be selected toelicit a measurable and reversible change in fluorescence.

The methods of the present invention enable the identification of manydifferent types of compounds capable of modulating mitochondrialfunction. For example, compounds that impact energy output generally,e.g., glycolysis, oxidative phosphorylation, Krebs cycle, etc. can bedetected by the present methods. Additionally, mitochondrial function isaffected by the activity of many different channels and transporters inthe mitochondrial membrane, e.g., potassium pumps, transporters andchannels, proton pumps and proton channels. Compounds that affect theactivity of any of these channels and transporters can be detected bythe methods of the present invention.

The methods described herein are useful for identifying compounds thatcan be used to treat certain diseases and alter mitochondrial states.Compounds that increase mitochondrial respiration or other mitochondrialoutput can be identified by the present methods by testing compounds forincreasing mitochondrial respiration. Such compounds can be used toincrease the overall fitness of an animal or improve the condition of adiseased tissue. In addition, compounds that decrease the energy outputof the mitochondria and therefore decrease the energy available to cellscan be identified by the methods of the present invention. Compoundsthat decrease mitochondrial respiration or other mitochondrial outputare useful to decrease the division and spread of cancer cells. Inaddition, the present methods can be used to identify compounds that areprophylactic, e.g., to induce or mimic ischemic preconditioning ofcardiac muscle.

Preferred drugs used to induce or mimic ischemic preconditioning act byaffecting only mito_(KATP) channels. Not wishing to be bound by theory,in ischemic preconditioning it is believed that opening of mitochondrialK_(ATP) channels dissipates the inner mitochondrial membrane potentialestablished by the proton pump. This dissipation accelerates electrontransfer by the respiratory chain, and if uncompensated by increasedproduction of electron donors (such as NADH), leads to net oxidation ofthe mitochondria. The methods of the present invention include themeasurement of mitochondrial redox state by recording the fluorescenceof FAD-linked enzymes in the mitochondria, to selectively screen forcompounds that affect that redox state.

Preferred compounds for use on myocardial cells detected by the methodsof the present invention activate a mitochondrial K_(ATP) channel andreversibly oxidize the mitochondrial matrix, without having an effect onsarcolemmal K_(ATP) channels. As shown in Examples which follow,diazoxide is an example of such a compound that can be detected usingthe present methods. Diazoxide is a commonly used antihypertensive drugwhich causes dose-dependent mitochondrial oxidation and iscardioprotective. It is believed that diazoxide is an agonist ofmitoK_(ATP) channels. The simultaneous measurement of flavoproteinfluorescence using the methods of the present invention and sarcolemmalK_(ATP) currents (I_(KATP)) in intact rabbit ventricular myocytes, usingwhole-cell patch clamp, as described in the Examples below, show thatdiazoxide, a K_(ATP) channel opener, selectively activates mitochondrialK_(ATP) channels. Diazoxide also protects myocytes against simulatedischemia. Thus, the methods of the present invention are useful forscreening for compounds that are capable of protecting cells fromischemia.

The methods of the present invention can also be used to detectcompounds that block oxidation induced by other chemicals. In such amethods, the candidate compounds are potential oxidation blockers andare added before, during or after addition of compounds that induceoxidation of the mitochondria. For example, using methods of the presentinvention, it was found that 5-HD, which has been shown to inhibitK_(ATP) channels in sarcolemma (Notsu, T., et al., J. Pharmacol. Exp.Ther., 1992, 260: 702-708), and isolated mitochondria (Garlid, K. D., etal., Biophy. J., 1997, 72: A39 (Abstract)), reversibly blocks theflavoprotein oxidation induced by diazoxide (FIG. 2a). 5-HD is widelyused to block ischemic preconditioning and cardioprotection induced byK_(ATP) channel openers. 5-HD is an effective blocker of mitochondrialK_(ATP) channels.

As discussed above, it is believed that ischemic preconditioning is dueto activation of mitochondrial K_(ATP) channels. Thus, preferred assaysfor testing compounds that activate mitochondrial K_(ATP) channelsutilize a cellular ischemia model. In this model, cells are centrifugedinto a pellet to simulate the restricted extracellular space and reducedoxygen supply during ischemia, sampled at designated time points andstained with a hypotonic (85 mOsm) trypan blue solution to test theosmotic fragility of the membrane. (See Vander Heide, R. S., et al., J.Mol. Cell Cardiol, 1990, 22: 165-181). Previous studies have shown thatsimulated ischemia preconditions myocytes in this model, (Liu, Y., etal., Basic Res. Cardiol, 1996, 91:450-457; Armstrong, S., et al.,Cardiovasc. Res., 1994, 28: 72-77), and that the underlying mechanismsfor the protection are similar to those in intact hearts. (Armstrong,S., et al., Cardiovasc. Res., 1994, 28: 72-77). Drugs such as diazoxideprotect rabbit ventricular myocytes to the same extent aspreconditioning. Interestingly, a cardioprotective EC₂₅ of 11 μMdiazoxide has been reported in intact hearts. (Paucek P., et al., J.Mol. Cell. Cardiol., 1997, 29: A199(Abstract)). This concentrationcorresponds closely to that which was observed to induce flavoproteinoxidation, using the methods of the present invention (FIG. 5).

As also discussed above, it is believed that mitochondrial K_(ATP)channels may serve as effectors of cardioprotection by K_(ATP) channelopeners and protect myocytes against ischemic damage. It is theorizedthat this occurs through the dissipation of mitochondrial membranepotential resulting in a decrease in the driving force for calciuminflux through the calcium uniporter. It has been reported thatinhibition of the mitochondrial calcium uniporter by ruthenium redprotects hearts against ischemia and reperfusion injury, consistent withthis hypothesis. (Miyamae, M., et al., Am. J. Physiol, 1996, 271 :H1245-H2153; Figueredo, V. M., et al., Cardiovasc Res. 1991, 25 :337-342; Park, Y., et al., J. Pharmacol Exp. Ther., 1990, 253: 628-635).Another possibility is that opening of mitochondrial K_(ATP) channels,by decreasing the membrane potential, could promote the binding of theendogenous mitochondrial ATPase inhibitor IF₁, and thus conserve ATPduring ischemia. (Rouslin, W., J. Bioenerg. Biomembr., 1991, 23:873-888). Finally, a change of mitochondrial membrane potential couldalter glycolytic pathways during ischemia in favor of myocyte survival.

In some embodiments of this methods, the method further comprisesmeasuring mitochondrial K_(ATP) ion channel currents in the cells usedin the assay.

As discussed above, preferably the candidate compounds used on cells ofmyocardial origin do not substantially activate a sarcolemmal K_(ATP)channel. Sarcolemmal K_(ATP) ion channel currents (I_(KATP)) can bemeasured simultaneously with fluorescence in cells, e.g., ventricularcells, by methods known in the art, such as whole cell patch-clampmethod, which is generally preferred. See the examples for suitableprocedures for this method. References herein to a “standard whole-cellpatch assay” are intended to refer to the protcol described in theexamples below. Changes in channel currents can be measured in responseto the addition of drugs to the cells.

Depending on the intended use of the drug, different effects will bedesired. For example, preferred drugs that are to be used to inducecardioprotection do not substantially change the sarcolemmal K_(ATP) ionchannel currents. Preferably, compounds identified by the methods of theinvention include those that exhibit at least about a 100-fold greateractivation of mitochondrial K_(ATP) channels relative to activation ofsarcolemmal K_(ATP) channels, more preferably about a 500-fold greateractivation of mitochondrial K_(ATP) channels relative to activation ofsarcolemmal K_(ATP) channels, still more preferably about a 1000-foldgreater activation of mitochondrial K_(ATP) channels relative toactivation of sarcolemmal K_(ATP) channels.

As described above, an example of a use of this assay includes theidentification of compounds that induce or mimic ischemicpreconditioning by increasing oxidation of the mitochondria. Thus,preferably the methods of the present invention identify that increasemitochondrial oxidation.

As discussed above, the methods of the present invention can be used toidentify compounds that affect, e.g., the numerous transporters andchannels in mitochondria. While the methods have been described abovewith particular attention to mitochondrial K_(ATP) channels, the methodscan be applied to any channel or transporter. For example, to identifycompounds that affect proton channels, resulting in a change in theredox state of the mitochondria, the candidate compounds are contactedwith the test cells as described above. If the candidate compoundchanges the redox state of the mitochondria, such change will bedetected, e.g., by a change in fluorescence, and a mitochondrialmodulating compound will be identified. The effect of these compounds onthe redox state can be measured simultaneously with the effect of thecompounds on the activity of other channels or transporters through theuse of methods described herein, e.g., whole-cell patch clamp.

The compounds identified by the methods of the present invention areuseful as additions to enhance cell vitality in cell culture and invitro assays.

In certain embodiment of the present invention, the test cells comprisecells in a tissue of interest. In such a method, the tissue of interestis treated with the one or more candidate compounds ex vivo. In thisembodiment, the tissue of interest or cells from the tissue (sometimesknown as primary cells), is removed from a host organism. The cells arethen used in the methods described above. That is, the cells arecontacted with one or more candidate compounds and the change in themitochondria redox state of those cells is detected. Subsequently, thetreated cells, i.e., oxidized cells, can be implanted back into therecipient host organism.

In certain embodiments, the candidate compounds are contacted to thetest cells in vivo. The in vivo assays of the invention are particularlyuseful for subsequent evaluation of mitochondrial modulating compoundsexhibiting suitable activity in an in vitro assay. For example, ananimal model of cardiac muscle damage accompanying ischemia or aninvasive surgical procedure such as balloon angioplasty is useful. Onesuitable protocol involves administering to the animal a suitablevehicle or vehicle combined with one or more mitochondrial modulatingcompounds of interest. The amount of the mitochondrial modulatingcompound administered will vary depending on several parametersincluding the extent of damage associated with the ischemia or surgicalprocedure of interest. In instances where balloon angioplasty isemployed, the animal will typically receive a candidate compound in adose (e.g., i.m. or i.p.) of between about 0.5 to 100, preferably 1 to20 and more preferably about 10 mg/kg body weight of the animal. Apreferred dosage schedule provides for administration of the compoundstarting 24 hours prior to conducting an invasive surgical procedure orinducing ischemia. Daily injections, e.g., i.m. or i.p., of the compoundare generally preferred. Subsequently, the animals are euthanized andthe organ, e.g., heart removed for examination.

The term “invasive surgical procedure” means a medical or veterinarytechnique associated with significant damage to an organ such as theheart, liver or the kidney, or a limb. The invasive surgical procedurecan be associated with techniques involving, e.g., cardiac surgery,abdominothoracic surgery, arterial surgery, deployment of animplementation (e.g., a vascular stent or catheter), or endaterectromy.Preferably, the invasive surgical procedure is performed on a mammalsuch as a primate, particularly a human, rodent or a rabbit, or adomesticated animal such as a pig, dog or a cat. Ischemia can be inducedin the animal by methods known in the art.

In other embodiments, the compound is administered to the animal eitheras a sole active agent or in combination with other active compounds(e.g., 5-HD), or other candidate compounds to be tested. In mostembodiments, activity of the candidate compound in a given in vivo assayis compared to a suitable control (e.g., a sham-operated animal) inwhich the assay is conducted the same as the test assay but withoutadministering the compound to the test subject. A variety of testsubjects can be employed, particularly mammals such as rabbits,primates, various rodents and the like.

As noted above, the assays (either in vitro or in vivo) can be conductedin a wide variety of cells, tissues and organs. The assays can detectuseful mitochondrial modulating compounds by measuring the redox stateof the mitochondria in several cell, tissue and organ settings.

The present invention further provides in vitro kits for detectingcompounds capable of modulating mitochondrial function.

The invention also includes diagnostic kit formulations. Kits of theinvention preferably include test cells and medium for use in the assayin an immediately usable or readily reconstituted form and preferablyany other reagents necessary to ensure the activity and/or growth of thecells. Optionally, the kit includes a known mitochondrial oxidizing orreducing agent. Also optionally, the kit further contains a detectiondevice to facilitate determination of whether the candidate compound isa modulating compound. Also, the kit optionally contains multi-wellplates or test tubes for running the assay.

More particularly, in certain preferred kits of the invention, the kitincludes a vial or vessel containing test cells and an ampule or vialcontaining growth medium to sustain the test cells. Such a kit may alsoinclude photographic film or other detection device. In use, the testcells are mixed with the growth medium and the candidate compounds areadded. After a predetermined period of time, an aliquot of the assaymixture is spotted on or placed near the film, and the film isdeveloped. The degree of spotting on the film indicates the redox stateof the cells. Preferably a control, containing the test cells and growthmedium, but not the candidate compound, is run simultaneously. Forexample, if the candidate compound increases the oxidative state of themitochondria, the film containing the cells contacted with the candidatesample will have a larger and/or darker spot than the control. If thecandidate compound decreases the oxidative state, the control film willhave a larger and/or darker spot than the cells treated with thecandidate compound. The difference in the spotting on the film for themixture containing the candidate compound and the control is indicativeof a change in the redox state of the cells in response to the candidatecompound.

All documents mentioned herein are incorporated herein by reference intheir entirety.

The present invention is further illustrated by the following examples,which are provided to aid in the understanding of the invention and arenot construed as a limitation thereof.

General Comments of Examples

The following examples were performed according to with The Guide forthe Care and Use of Laboratory Animals published by the US NationalInstitutes of Health (NIH Publication No. 85-23, revised 1985).

Chemicals

In the following Examples, Collagenase (type II) was purchased fromWorthington. Diazoxide was obtained from Sigma Chemical Co. Pinacidiland 5-hydroxydecanoic acid sodium (5HD) were purchased from ResearchBiochemical Int. Tetramethylrhodamine ethyl ester (TMRE) was obtainedfrom Molecular Probes. Diazoxide, pinacidil and TMRE were dissolved inDMSO before added into experimental solutions. The final concentrationof DMSO was less than 0.1%.

Electrophysiology and Flavoprotein Fluorescence Measurement

In the following Examples, ventricular myocytes were isolated from adultrabbit hearts by conventional enzymatic dissociation, according to Liu,Y., et al., “Synergistic modulation of ATP-sensitive K⁺ currents byprotein kinase C and adenosine: implications for ischemicpreconditioning”, Circ. Res., 1996, 78: 443-454, then washed severaltimes with calcium-free solution. Calcium concentration was graduallybrought back to 1 mM. Cells were then cultured on laminin-coatedcoverslips in M199 culture medium with 5% fetal bovine serum at 37° C.Experiments were performed over the next two days.

For whole-cell patch recordings, the internal pipette solution contained(in mM): 120 K-glutamate, 25 KCI, 0.5 MgCl₂, 10 K-EGTA, 10 HEPES and 1MgATP (pH 7.2 with KOH). The external solution included (in mM): 140NaCl, 5 KCI, 1 CaCl₂, 1 MgCl₂, and 10 HEPES (pH 7.4 with NaOH).Whole-cell currents were elicited every 6 sec from a holding potentialof −80 mV by two consecutive steps to −40 mV (for 100 msec) and 0 mV(for 380 msec). Currents at 0 mV were measured 200 msec into the pulse.Endogenous flavoprotein fluorescence was excited using a xenon arc lampwith a bandpass filter centered at 480 nm, but only during the 100 msecstep to −40 mV t to minimize photobleaching. Emitted fluorescence wasrecorded at 530 nm by a photomultiplier tube and digitized (Digidata1200, Axon Instruments). (See Paucek, P., et al., “Cardioprotectiveeffect of diazoxide and its interaction with mitochondrial K_(ATP):possible mechanism of cardioprotection”, J. Mol Cell Cardiol, 1997, 29:A199(Abstract)). Relative fluorescence was averaged during theexcitation window and calibrated using the values after dinitrophenol(DNP) and sodium cyanide (CN) exposure.

In some cells, contracture occurred before the fully reduced level(after CN exposure) could be determined. In these cells, data wereexpressed as a percentage of the DNP-induced fluorescence, since thebasal redox state was nearly fully reduced (average 5% oxidation, n=5).

Flavoproteinfluorescence and Mitochondrial Imaging

In the following Examples, Confocal images were obtained using a Diaphot300 inverted fluorescence microscope with a PCM-2000 confocal scanningattachment (Nikon, Inc.). Fluorescence was excited by the 488 nm line ofan argon laser and the emission at 505-535 nm was recorded. A timeseries of images was collected at intervals of ˜10 sec and baseline,diazoxide, DNP and CN images were enhanced by averaging 8-10 sequentialimages having stable mean fluorescence intensities during the exposureto each agent.

To localize mitochondria, cells were loaded with 100 nM TMRE, whichdistributes into negatively charged cellular compartments, for 10 min.,(Lemasters, J. J., et al., “Measurement of electrical potential, pH, andfree calcium ion concentration in mitochondria of living cells by laserscanning confocal microscopy”, in Anonymous: Methods in Enzymology., NewYork, Academic Press, 1995, pp 429-417). TMRE fluorescence was excitedwith the 535 nm line of a helium neon laser and recorded at greater than605 nm. A pseudocolor palette was applied to visualize the relativeincrease in mitochondrial flavoprotein oxidation state.

Images were analyzed on a personal computer using the software programImageTool (University of Texas Health Sciences Center in San Antonio).All the recordings were performed at room temperature (21-22° C.).

Simulated Ischemia and Cellular Injury

In the following Examples, the procedure to determine cell injury wasmodified from Vander Heide, et al. Vander Heide, R. S., et al., “An invitro model of myocardial ischemia utilizing isolated adult ratmyocytes”, J. Mol. Cell Cardiol, 1990, 22: 165-181. After cellisolation, cells were washed with incubation buffer (in mM): NaCl 119,NaHCO₃ 25, KH2PO₄ 1.2, KCl 4.8, MgSO₄ 1.2, HEPES 10, glucose 11, andsupplemented with creatine, taurine and amino acids (pH 7.4). Calciumwas added into the buffer stepwise (0.25 mM every 5 minutes) to a finalconcentration of 1 mM. An aliquot of each cell suspension (0.5 ml) wasplaced in a 0.5 ml microcentrifuge tube and centrifuged for 20 secondsinto a pellet. Each pellet occupied a volume of about 0.2 ml.Approximately 0.25 ml of excess supernatant was removed to leave a thinfluid layer above the pellet, and 0.2 ml of mineral oil was layered onthe top of the pellet to exclude gaseous diffusion. After 60 min and 120min of pelleting, 5 μl of cell pellet were sampled through the oil layerand mixed with 75 μ1 of 85 mOsm hypotonic staining solution (in mM):NaHCO₃ 11.9, KH₂PO₄ 0.4, KCl 2.7, MgSO₄ 0.8, CaCl₂ 1 and 0.5%glutaraldehyde, 0.5% trypan blue.

Microscopic examination was performed 2-5 min after mixing to determinethe permeability of the cells to trypan blue. Cells permeable to trypanblue were counted as dead and expressed as a percentage of the totalcells counted (>200 for each sample).

The killing of cells by ischemia was quantified as percentage of thevital cells at the beginning of each experiment (78-90% of total,mean=82±1% n=24). The small percentage of cells (˜18%) that werenon-viable at the beginning of the experiment were mostly rounded andhad been damaged as a known consequence of the enzymatic isolationprocess. Mitra, R., et al., “A uniform enzymatic method for dissociationof myocytes from hearts and stomachs of vertebrates”, Am. J. Physiol,1985, 249: H1056-H1060.

EXAMPLE 1

Individual experiments in each of four groups were performed on cellsisolated from different hearts. In the control group (Cont), cells werepelleted and sampled at the 60 min and 120 min. For thediazoxide-treated group (Diazo), 50 μM of diazoxide was added into thesolution 15 min before the pelleting. In the third group (5-HD), 100 μMof 5-HD was added into the cell suspension 20 min prior to pelleting.Cells in the Diazo+5HD group were treated the same as in the third groupexcept that 50 μM of diazoxide was added into the cell suspension 15 minbefore pelleting. Once applied, drugs were not washed out, and thus werepresent throughout the period of simulated ischemia.

Data are presented in the Figures as means±SEM, and the number of cellsor experiments is shown as n. Analysis of variance (ANOVA) combined withTukey's HSD post-hoc test was used to test for differences among groupsfor electrophysiological and fluorescence data. Cell pelleting data wereanalyzed by two-way ANOVA combined with Tukey's HSD post-hoc test.p<0.05 was considered significant.

FIG. 1 shows results from simultaneous measurements of flavoproteinfluorescence and membrane I_(KATP) in cells exposed to diazoxide. Theperiods of drug treatment are marked with horizontal bars. Diazoxide(100 μM) induced reversible oxidation of the flavoproteins (FIG. 1A) butdid not activate I_(KATP) (FIG. 1B).

The redox signal was calibrated by exposing the cells to DNP followed byCN at the end of the experiments. DNP, induced maximal oxidation, whileCN caused complete reduction of the flavoproteins (FIG. 1A and FIG. 2A).Although membrane currents were unchanged by diazoxide, I_(KATP)eventually turned on after prolonged exposure to DNP (FIGS. 1B and 2B),indicating that these channels are operable under these experimentalconditions despite the inability of diazoxide to open them.

Diazoxide (100 μM, DIAZO(1)) reversibly increased mitochondrialoxidation to 48±3% of the DNP value (FIG. 3). FIG. 3 shows DIAZO(1),first exposure to diazoxide; DIAZO+5-HD(100), diazoxide in the presenceof 100 μM 5-HD; DIAZO+5-HD(500), diazoxide in the presence of 500 μM5-MD and DIAZO(2), second exposure to diazoxide. DNP, exposure todinitrophenol. The bar indicates the periods when cells were exposed todrug. The results show that this oxidation was reproducible, becauseafter washout of the response a second exposure to diazoxide (DIAZO(2))in the same cells increased flavoprotein oxidation to 43±5%. 5-HD (100μM) attenuated the oxidative effect of diazoxide by about half(DIAZ0+5HD(100)), while 500 μM 5-HD further reduced oxidation to 8±3%(DIAZ0+5-HD(500); p<0.01 vs. DIAZO(1), DIAZ0(2) and DIAZ0+5-HD(100)groups).

Treatment with diazoxide and 5-HD did not activate I_(KATP), whileprolonged exposure (>6 min) to DNP did turn on I_(KATP) (FIG. 4). InFIG. 4, DIAZO(1) is the first exposure to diazoxide, DIAZ0+5-HD(100)means diazoxide in the presence of 100 μM 5-HD, DIAZO+5-HD(500) meansdiazoxide in the presence of 500 μM 5-MD and DIAZ0(2) means secondexposure to diazoxide. DNP indicates exposure to dinitrophenol. The barindicates the periods when cells were exposed to drug.

The EC₅₀ for diazoxide to induce mitochondrial oxidation is 27 μM, asshown in FIG. 5. Each point in FIG. 5 constitutes measurements from 5-6cells. *=p<0.01 vs. DIAZ0(1), DIAZ0(2) and DNP groups.

EXAMPLE 2 Effect of Pinacidil on Redox State

Another K_(ATP) opener, pinacidil, which opens sarcolemmal K_(ATP)channels and is known to induce pharmacological preconditioning. Critz,S., et al., “Pinacidil but not nicorandil opens ATP sensitive K⁺channels and protects against simulated ischemia in rabbit myocytes”, J.Mol Cell Cardiol, 1997, 29: 1123-1130.

As shown in FIG. 6A, pinacidil (100 μM) induced 35±8% mitochondrialoxidation, comparable to the effect of diazoxide exposure in the samecell (41±5%). Unlike diazoxide, pinacidil activated sarcolemnal I_(KATP)(0.74±0.54 nA measured at 0 mV) in addition to inducing flavoproteinoxidation, (FIG. 6B) suggesting that pinacidil activates bothmitochondrial and sarcolemmal K_(ATP) channels.

EXAMPLE 3

The subcellular site of diazoxide action was localized by imagingflavoprotein fluorescence (FIG. 8). Fluorescence is low under controlconditions (Control), but exposure to diazoxide (Diazo) increasedfluorescence in strips parallel to the myofibril orientation. Subsequentexposure to DNP increased fluorescence even further (DNP), in a patternsimilar to that revealed by diazoxide. CN reduced the fluorescence tothe basal level (CN). The distribution of fluorescence induced bydiazoxide and DNP is as expected for mitochondria, which occupy ˜35% ofcardiomyocyte volume and are clustered longitudinally betweenmyofibrils. Sommer, J. R., et al., “Ultrastructure of cardiac muscle, inBerne RM (ed)”, Handhook of Physiology. Section 2. The CardiovascularSystem. Bethesda, Md., Am. Physiol Soc, 1979, pp 113-186.

This correspondence was further confirmed by using TMRE (old FIG. 3B),which distributes into negatively charged cellular compartments, tolocalize mitochondria. Lemasters, J. J., et al., “Measurement ofelectrical potential, pH, and free calcium ion concentration inmitochondria of living cells by laser scanning confocal microscopy, inAnonymous: Methods in Enzymology. New York, Academic Press, 1995, pp429-417. The pattern of TMRE fluorescence was virtually identical tothat of the flavoprotein fluorescence induced by diazoxide.

EXAMPLE 4

FIG. 9 shows the fraction of cells killed by 60 or 120 min of ischemiaas a percentage of the total number of viable cells prior to ischemia.Cell killed (%) was calculated as number of cells killed by ischemia asa percentage of the total viable cells prior to ischemia. In that FIG.9, the following designations have the following meanings: Cont,control. Diazo, 50 μM diazoxide. 5-HD, 100 μM 5-HD. Diazo+5-HD,diazoxide in the presence of 5-HD. *=p<0.01 vs. the other three groups.60 min and 120 min killed 35±2% and 46±4% of cells respectively in thecontrol (Cont). However, inclusion of 50 ±M diazoxide significantlydecreased cell death during simulated ischemia to about half of that inthe controls (18±3% after 60 min, and 23±4% after 120 min, p<0.01 vs.Cont). The protection by diazoxide was completely blocked by 100 μM 5-HD(31±2% after 60 min and 47±2% after 120 min). 5-HD alone did notsignificantly alter the percentage of cells killed by simulatedischemia: 31±2% after 60 min and 47±2% after 120 min. Glybenclamide (1μM) also blocked the protection from diazoxide (data not shown).Diazoxide at 100 μM had a similar protective effect (data not shown).

For each experiment, there was always an isochronal nonischemic group inwhich cells were not pelleted. In these groups, less than 5% oftrypan-blue-resistant cells became permeable to trypan blue during the2-hour experiments.

The invention has been described in detail with particular reference tothe preferred embodiments thereof. However, it will be appreciated thatmodifications and improvements within the spirit and teachings of theinventions may be made by those in the art upon considering the presentdisclosure.

What is claimed is:
 1. A method for identifying a compound capable ofincreasing or decreasing the redox state of mitochondria, comprising:(a) contacting a eukaryotic cell with one or more candidate compounds,and (b) measuring fluorescence of an endogenous mitochondrial component;(c) detecting a change in fluorescence upon contact with a candidatecompound, wherein a change in fluorescence is indicative of an increaseor decrease in the mitochondrial redox state of the cell; and (d)selecting a compound or compounds which causes a change in fluorescence,wherein the candidate compound activates a mitochondrial K_(ATP)channel.
 2. The method of claim 1 wherein the candidate compound doesnot substantially activate a sarcolemmal K_(ATP) channel.
 3. A methodfor identifying a compound capable of increasing or decreasing the redoxstate of mitochondria, comprising. (c) contacting a eukaryotic cell withone or more candidate compounds, and (b) measuring fluorescence of anendogenous mitochondrial component; (c) detecting a change influorescence upon contact with a candidate compound, wherein a change influorescence is indicative of an increase or decrease in themitochondrial redox state of the cell; and (d) selecting a compound orcompounds which causes a change in fluorescence; wherein the oxidationstate of the endogenous mitochondrial flavin moiety is monitored byfluorescence, and an increase in fluorescence indicates a increasedoxidation state and a reduced fluorescence indicates an reducedoxidations.